Project Summary The principal goal of this research is to address the urgent need for novel, improved therapeutics for the treatment of fungal infections caused by Candida albicans (Ca). Invasive or chronic infection from Ca is a major, growing, public health concern, and mortality due to fungal diseases currently exceeds that from both malaria and tuberculosis. Invasive Candida mortality is as high as 67% in some patient populations. Drug discovery research for new anti-infective agents has failed for decades to meet the demands for treatment of invasive yeasts and fungi, especially for drug-resistant pathogens. We propose an innovative strategy that seeks to discover new anti-Ca natural products from understudied sources, using genetic methods to enhance metabolite production, and a new analytical technique to enrich discovery. The three overarching objectives of this research project include: 1) Identification of novel compounds that circumvent resistance mechanisms, 2) Identification of novel compounds with broad-spectrum activity against Ca and 3) Establishment of a workflow for application to other fungal pathogens. The foundation of the project is understudied natural products (NP) sources. Natural products are the primary origin of antibiotics: In the 33 years between 1981-2014, 82 new antibiotics were registered with the FDA as either native NP or NP derivatives, while only 29 totally synthetic compounds were discovered. Indeed, 2 of the 3 classes of antifungal drugs are NPs (polyenes and echinocandins). Despite decades of study, there are perhaps 250,000 NP known, a surprisingly small number compared to estimates of global microbial biodiversity of over 10 M species. Taken with post-genomic-era discovery of silent biosynthetic pathways, the number of genetically-encoded NP yet to be discovered must dwarf those already known. NP studied in our program will be sourced from endophytic fungi found within mangrove tissues. Mangroves have recently been described as a ?hot spot? of fungal diversity yet US populations are unstudied. We use epigenetic manipulation to induce expression of silent fungal natural product biosynthetic pathways to reveal untapped chemodiversity. Our preliminary data highlight the power of this approach to discover Ca-active compounds that would have been missed using traditional culture methods. Our project brings further innovation in culture miniaturization that maximizes screening throughput, and a chromatographic technique to reduce effort lost in chemotype re- discovery. Active NPs brought forward in our workflow, prioritized based on potency and selectivity over mammalian cytotoxicity, will accumulate advanced biological and pharmacological profiling, including spectrum of activity, biofilm inhibition, mode of action and drug-like properties. Based on the success of this R21 in identifying potent and selective inhibitors of Candida albicans, future hit- to-lead development of novel candidate therapeutics or molecular tools will be pursued, as well as application of the screening workflow to other problematic fungal infections, such as Aspergillus, and Cryptococcus.